Immobilization of biopolymers to aminated substrates by direct adsorption

ABSTRACT

An assay article for detection biopolymers contained in a sample is described. The assay article includes a substrate and a biopolymer directly adsorbed on the surface of the substrate. A plurality of biopolymers may be adsorbed on the surface of the substrate to form an array. Also disclosed is a method of making the assay article. In the preferred method, an aminated polypropylene substrate is used. A biopolymer is contacted with the aminated substrate under a condition sufficient for direct adsorption of the biopolymer on the surface of the substrate. A method of detecting a target biopolymer contained in a sample is also disclosed. In this method, a substrate is contacted with either a probe or target biopolymer under a condition sufficient for a direct adsorption of either the probe or target biopolymer on the substrate to form a probe assay article or a target assay article. Then, the probe assay article is contacted with the target biopolymer, or the target assay article is contacted with the probe biopolymer under a condition that allows the formation of a probe-target complex. Finally, the complex is detected and the presence of the complex is used as a measurement for the presence or the amount of the biopolymer target contained in the sample.

AREA OF THE ART

The present invention relates generally to solid substrates withimmobilized biopolymers. In particular, the invention relates toaminated substrates with adsorbed biopolymers, methods of theirconstruction and methods of their use in detection of targetbiopolymers.

DESCRIPTION OF THE PRIOR ART

Analysis of unknown biopolymers targets often involves their specificbinding to known biopolymers probes. The most common technique employingimmobilized biopolymers is the Southern blot hybridization technique, inwhich a set of DNA targets is immobilized on a membrane and a solutioncontaining labeled DNA probe molecules is used to bathe the membraneunder conditions where complementary molecules will anneal. In ananalogous technique called Northern blot hybridization, RNA targets areimmobilized on membranes and anneal to complementary RNA probes. Reverseblot hybridization employs the opposite approach. Instead ofimmobilizing DNA targets, a set of DNA probes is immobilized on a solidsurface and the unknown labeled DNA target is present in the liquidphase.

Arrays, constructed by attaching a plurality of the same or differentbiopolymers to discrete isolated areas on the surface of the substrate,are becoming increasingly important tools in analysis of unknownbiopolymers, such as gene expression analysis, DNA sequencing, mutationdetection, polymorphism screening, linkage analysis, genotyping singlenucleotide polymorphisms (SNPs), and screening for alternative splicevariants in gene transcripts.

Gene expression analysis is a method of critical importance to basicmolecular biological research. Since in higher organisms, the choice ofgenes being expressed in any given cell has a profound effect on thenature of the cell, gene expression analysis can provide a key todiagnosis, prognosis, and treatment of a variety of diseases in animals,including humans and plants. Additionally, gene expression analysis canbe used to identify differentially-expressed novel genes, to correlate agene expression to a particular phenotype, to screen for a diseasepredisposition, and to conduct toxicity testing.

Typically, in the gene expression analysis, an array of probe nucleicacids is formed by attaching a set of individual gene-specific probes toa solid substrate in a regular pattern, so that the location of eachprobe is known. The array is contacted with a sample containing targetnucleic acids under hybridization conditions. The hybrids are detectedusing a wide variety of methods, most commonly by employing radioactiveor fluorescent labels.

There are two general methods of forming polynucleotide arrays for thegene expression analysis. The first method involves in situ synthesis ofoligonucleotides in predetermined positions of a solid substrate. Thesecond method includes the association of pre-synthesizedoligonucleotides or polynucleotides with a solid substrate. In situsyntheses of oligonucleotides on glass modified with an aliphaticpolyether linker (Southern, E. M. et al., Genomics 13:1008, 1992) andpolypropylene as a solid substrate (U.S. Pat. No. 5,554,501) have beenreported. The in situ method, however, suffers from certainshortcomings. For example, since the addition of each nucleotide is aseparate reaction, the reproducibility and reaction yield may varywidely between different locations on the substrate, as well as betweendifferent substrates. Consequently, it is hard to obtain accurate andreproducible data with arrays formed by direct synthesis.

Alternatively, pre-synthesized polynucleotides may be immobilized on asubstrate by ultraviolet cross-linking, chemical adhesion, or covalentbonding. Typically, a polynucleotide, a solid substrate, or both, arederivatized to initiate such immobilization. Usually it is preferredthat the solid substrate is capable of immobilizing probe DNA, whilebeing substantially inert to any other DNA. Glass is a commonly usedsolid substrate, because it is inexpensive and of good optical quality.Various types of pre-derivatized glass substrates are commerciallyavailable, including microscope slides coated with poly-L-lysine oramino propyl silane, or glass slides with exposed aldehydefunctionalities. However, the derivatization of a glass surface createsa positive electrostatic net charge, which results in undesirablenon-specific electrostatic binding of nucleic acids to the solidsubstrate during subsequent hybridization procedures. Numerous othersurface coatings for efficient immobilization of polynucleotides havebeen proposed and include an isolate of the naturally occurring musseladhesive protein (U.S. Pat. No. 5,024,933), nucleoside phosphate (U.S.Pat. No. 4,818,681), and salt or cationic detergent (U.S. Pat. No.5,610,287), to name a few. These methods, however, also entailunspecific binding of nucleic acids on the substrates. Such unspecificbinding of DNA makes interpretation of the hybridization resultsdifficult.

Polynucleotides themselves can be derivatized prior to the binding to asolid substrate. For example, U.S. Pat. No. 6,048,695 describesepoxide-modified DNA which is readily affixed to an unmodified solidsubstrate, such as an underivatized glass surface. Finally, both apolynucleotide and a solid substrate may be modified to allow efficientcovalent bonding. For example, U.S. Pat. No. 5,215,882 disclosesmodifying a nucleic acid with a primary amine or equivalent, followed bythe reaction of the modified nucleic acid with the solid substratehaving free aldehyde groups in the presence of a reducing agent.However, the derivatization of polynucleotides and their subsequentcovalent binding to solid substrates are long and tedious processes.Consequently, the arrays produced by conventional techniques are fairlyexpensive ($6-$36 per slide). Similar problems exist in respect toimmobilization of other biopolymers on solid substrates.

In summary, the conventional immobilization methods do not providedesirable fast hybridization and high specificity of binding of targetsto probes. Additionally, currently available methods of manufacturingassay articles for use in biopolymer detection are slow, tedious, andeconomically unfavorable.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide acost-efficient, rapid and convenient method of making an assay articleand a method of using such an assay article for a biopolymer detection.Particularly, it is an object of the present invention to develop amethod of making an assay article by direct adsorption of biopolymers onsolid substrates.

The present invention is based on the discovery that modifiedsubstrates, and particularly plasma aminated polypropylene substratesare capable of direct and stable adsorption of nucleic acids, proteins,polypeptides, and their analogues without chemical crosslinking.Consequently, one aspect of the present invention provides a method ofmaking an assay article for use in biopolymer detection. The methodcomprises the steps of:

-   -   (a) providing a biopolymer;    -   (b) providing an aminated substrate; and    -   (c) contacting the biopolymer with a surface of the aminated        substrate under a condition sufficient for a direct adsorption        of the biopolymer on the surface of the substrate.

According to embodiments of the present invention, the substrate may befabricated in a form of foams, filaments, threads, sheets, films,slides, gels, membranes, beads, plates, and like structures. In oneembodiment of the present invention, the step of providing thebiopolymer includes providing a solution of the biopolymer; and the stepof contacting the biopolymer comprises:

-   -   (a) placing an aliquot of the biopolymer solution on the        substrate; and    -   (b) air-drying the substrate to directly adsorb the biopolymer        on the surface of the substrate.

In accordance with one embodiment of the present invention, a pluralityof biopolymers may be placed and adsorbed on the surface of the aminatedpolypropylene substrate in an array.

Another aspect of the present invention provides a method of detecting atarget biopolymer contained in a sample. The method comprises the stepsof:

-   -   (a) providing an aminated substrate;    -   (b) providing a probe biopolymer that can form a complex with        the target biopolymer;    -   (c) contacting either the probe or target biopolymer with a        surface of the aminated substrate under a condition sufficient        for a direct adsorption of either the probe or target biopolymer        on the substrate surface to form a probe assay article or a        target assay article, respectively;    -   (d) contacting the probe assay article with the target        biopolymer, or contacting the target assay article with the        probe biopolymer under a condition that allows the formation of        a complex comprising the probe and the target biopolymers; and    -   (e) detecting and determining the presence of the complex as a        measurement for the presence or the amount of the target        biopolymer contained in the sample.

The complex of the probe and the target biopolymers may also include areporter. The reporter may be selected from a group consisting of: dyes,chemiluminescent compounds, enzymes, fluorescent compounds, metalcomplexes, magnetic particles, biotin, hapten, radio frequencytransmitters, and radioluminescent compounds. An assay article may beformed by attaching the same or different unmodified probes or targetbiopolymers to discrete areas of the substrate to produce an array. Thesignal produced by the report molecules immobilized on the array may bedetected and recorded by a number of means, such as a laser with aconfocal array reader, phosphor imager, or a CCD camera.

A further aspect of the present invention provides an assay article fordetecting biopolymers. The assay article comprises a substrate and abiopolymer directly adsorbed on the surface of the substrate. In oneembodiment, a plurality of the same or different biopolymers is attachedto discrete, isolated areas of the substrate by direct adsorption toform an array.

Another aspect of the present invention provides a test kit fordetecting a target biopolymer contained in a sample. The kit comprisesan aminated polypropylene substrate and a probe biopolymer directlyadsorbed on a surface of the substrate. When the probe polymer iscontacted with the target biopolymer, they form a complex that can bedetected by utilizing reporters and signal detection devices.

The present invention is well-suited for use in creating biopolymerarrays and polynucleotide arrays, such as gene expression micro-arraysfor use in gene expression analysis, in particular. The polynucleotidearrays may be used for the evaluation or identification of biologicalactivity. The present invention may also be used in creatingpolynucleotide arrays for the purpose of polynucleotide sequencing.Further, the assay articles of the present invention may contain a rangeof adsorbed biopolymers and may be utilized in hybridization assays andimmunoassays.

The present invention provides many advantages. Since the inventionallows for the adsorption of biopolymers directly on a solid substratewithout chemical crosslinking, costly production of modifiedbiopolymers, such as thiol- or amino-modified DNA, may be avoided. Also,the task of making arrays is greatly simplified and the production costsare significantly reduced, because the biopolymers are simply air-driedon the substrate.

DESCRIPTION OF THE FIGURES

The above-mentioned and other features of this invention and the mannerof obtaining them will become more apparent, and will be best understoodby reference to the following description, taken in conjunction with theaccompanying FIG. 1.

FIG. 1 shows the hybridization results of labeled target cDNA fromactin, β-microglobulin, G3PDH, and p53 with their corresponding probecDNA immobilized on polypropylene substrates. TNF-α immobilized on thesubstrate was used as a control for a non-specific hybridization.

FIG. 2 shows attachment of human IgG to aminated polypropylene support.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a method of making an assayarticle for use in biopolymer detection. The method comprises the stepsof:

-   -   (a) providing a biopolymer;    -   (b) providing an aminated substrate; and    -   (c) contacting the biopolymer with a surface of the aminated        substrate under a condition sufficient for a direct adsorption        of the biopolymer on the surface of the substrate.

The term “biopolymer” as used herein refers to nucleic acids,polynucleotides, polypeptides, proteins, and analogues thereof. As usedherein, “polynucleotide” refers to a polymer of deoxyribonucleotides orribonucleotides, in the form of a separate fragment or as a component ofa larger construction. “Polynucleotide”, as used herein, may be DNA,RNA, or a DNA analog, such as PNA (peptide nucleic acid). The DNA may bea single- or double-stranded DNA, or a DNA amplified by PCR technique.The RNA may be a mRNA. The length of the polynucleotides may be fromabout 20 bp to about 10 kb. In accordance with one embodiment of thepresent invention, the polynucleotide is a complementary DNA (cDNA). Thelength of a cDNA polynucleotide may be in the range of about 100 bp toabout 10 kb, preferably, 200 bp to 1000 bp.

As used herein, “polypeptide” refers to a polymer of amino acids,wherein the α-carboxyl group of one amino acid is joined to the α-aminogroup of another amino acid by a peptide bond. A protein may compriseone or multiple polypeptides, linked together by disulfied bonds.Examples of the protein include, but are not limited to, antibodies,antigens, ligands, receptors, etc.

It is a discovery of the present invention that biopolymers may beattached to substrates by direct adsorption and without any chemicalcross-linking with the substrate. We found that polypropylene substratesare particularly useful for direct adsorption. The direct adsorption isfurther improved by modifying the substrates prior to contacting themwith biopolymers. The substrates may be modified by introducing afunctionality selected from a group consisting of: amino, carboxyl,thiol, and their derivatives. In one embodiment, the substrate ismodified by introduction of an amine group.

The methods for introduction of amine groups onto the polypropylenesurface are described in the commonly assigned U.S. Pat. No. 6,013,789,the relevant content of which is incorporated herein in its entirety byreference. In short, amino groups may be introduced onto the surface ofa polypropylene medium by using a plasma discharge in an ammonia- ororganic-amine-containing gas. The “plasma” is most preferably an ionizedgas, which gains sufficient ionization energy from an electromagneticfield.

Preferably, the ionization energy is applied by a radio-frequency plasmadischarge, a microwave frequency plasma discharge, or a coronadischarge. In a particularly preferred embodiment of the invention, theamine is derived from an ammonia gas and the elevated energy state isachieved via radio-frequency plasma discharge. The aminatedpolypropylene is then utilized for direct adsorption of apre-synthesized biopolymer.

In order to accommodate a number of different testing techniquesincluding specialized testing equipment, aminated polypropylenesubstrates may be molded into any of a variety of shapes and forms.Examples of such shapes and forms of the aminated polypropylenesubstrates include, but are not limited to, foams, filaments, threads,sheets, films, slides, gels, membranes, beads, plates, and likestructures. An aminated polypropylene substrate may be fabricated in theform of a planar device having discrete isolated areas in the form ofwells, troughs, pedestals, hydrophobic or hydrophilic patches, die-cutadhesive reservoirs or other physical barriers to fluid flow. Examplesof such a substrate include, but are not limited to, a microplate or thelike. Because the substrate of the present invention is particularlyuseful in the preparation of biopolymer arrays for the evaluation oridentification of biological activity, the aminated polypropylenesubstrate is preferably in the form of a device having at least one flatplanar surface. Examples of such devices with flat surfaces include, butare not limited to, slides, sheets, films, or the like.

The size of the substrate can vary and depends upon the final use of theimmobilized biopolymers. Those skilled in the art will appreciate thatarrays of biopolymers immobilized on miniaturized solid supports havebeen under development for many years. These solid supports can bemeasured in terms of mm² planar surface area and can have numerousdifferent immobilized biopolymers, each attached to a differentsite-specific location on the miniaturized solid support. Solid supportsin the form of dipsticks and slides are also within the scope of thepresent invention. As known in the art, dipsticks typically arerectangular in shape with each side measuring a few centimeters.

Since the instant methods of assay article formation involve directadsorption of biopolymers on substrates, no chemical modification tobiopolymers is required. Here, the term “direct adsorption” meansadsorption without any chemical linkers. Unlike the related art, whichuses chemical crosslinking of biopolymers to the substrates, the presentinvention allows immobilization of both unmodified and modifiedbiopolymers on substrates by simple air-drying on the substrate. For thepurpose of the present invention, “unmodified biopolymer” means nativebiopolymer, and “modified biopolymer” means a biopolymer with introducedfunctional groups. For example, a modified biopolymer may bebiotinylated or aminated DNA.

In present invention, a biopolymer is immobilized on a substrate bycontacting the biopolymer with the substrate under a conditionsufficient for a direct adsorption of the biopolymer to the substrate. Acondition is sufficient if it allows the biopolymer to become adsorbedon the surface of the substrate in a stable way. While not wanting to bebound by the theory, it is believed that, under the conditions of thepresent invention, biopolymers may be adsorbed on a substrate by ionicand hydrophobic interaction.

For the purpose of the present invention, it is not crucial whichparticular method is used to carry out the step of contacting thebiopolymer with the substrate. In accordance with embodiments of thepresent invention, the contacting step may be carried out by jetprinting, solid or open capillary device contact printing, microfluidicchannel printing, silk screening, and printing using devices based uponelectrochemical or electromagnetic forces. For example, thermal inkjetprinting techniques utilizing commercially available jet printers andpiezoelectric microjet printing techniques, as described in U.S. Pat.No. 4,877,745, may be utilized to spot polynucleotides to the aminatedsubstrates. A Biomek High Density Replicating Tool (HDRT) (BeckmanCoulter, Calif.) may also be used for an automatic gridding.Alternatively, the contacting step may be carried out by manual spottingof the biopolymers on the aminated substrate. Examples of manualspotting include, but are not limited to, manual spotting with apipettor. It should be understood that the aminated substrate of thepresent invention may be exposed to biopolymers by any methods as longas the biopolymers are put in direct contact with the substrate.

In accordance with embodiments of the present invention, the step ofproviding the biopolymer may include providing a solution of thebiopolymer. The step of contacting the biopolymer with aminatedsubstrate may include:

-   -   (a) placing an aliquot of the biopolymer solution on the        substrate; and    -   (b) air-drying the substrate to directly adsorb the biopolymer        on the surface of the substrate.

The solution of the biopolymer may be any solution that delivers thebiopolymer to the surface of the substrate. Preferably, the solvent isan aqueous buffer having a pH from about 4 to about 13. In oneembodiment, the biopolymer is a polynucleotide, and a solution of thepolynucleotides in 50 mM sodium bicarbonate, pH 9, is spotted on theaminated substrate.

The concentration of biopolymers contained in aqueous solutions mayvary, depending on the type of molecule, the molecule size, the moleculestructure, and other factors that may influence solubility of themolecules. Preferably, the amount of the biopolymers applied to thesubstrate ranges from about 10⁻²⁰ to about 10⁻¹⁴ moles. For example, inone embodiment, the biopolymer is a polynucleotide, and the amount ofthe polynucleotide applied to the substrate is about 10⁻¹⁸ moles. Thesize of the biopolymer solution aliquot is not crucial, as long as itprovides sufficient amount of the biopolymer. Consequently, the size ofthe aliquots applied to the aminated substrate may vary, depending onthe concentration of the biopolymer in the solution and the assay needs.For example, the aliquot may be from about 0.1 nL to about 500 nL. Inone embodiment, the biopolymer is a polynucleotide, and aliquots ofabout 10 nL of the 1 nM polynucleotide solutions are placed on theaminated substrate.

In accordance with the present invention, the air-drying step isconducted for a period of time sufficient to allow adsorption of thebiopolymer solution. The length of the air-drying time depends on thevolume of the aliquots applied to the substrate, room temperature andhumidity. For micro- and nanoliter aliquots, the air-drying step maytake from about 5 to about 60 minutes. For example in one embodiment, 10nL aliquots are placed on the surface of the aminated substrate anddried at 22° C. for one hour or for about fifteen minutes at 35° C.

As mentioned above, many applications for utilizing immobilizedbiopolymers require biopolymers to be immobilized at site-specificlocations on a substrate surface. Accordingly, in the present invention,a plurality of biopolymers may be placed and adsorbed on the surface ofthe aminated polypropylene substrate in an array. In order to prepareordered arrays of biopolymers with each biopolymer located atsite-specific locations, including grids and 1×n arrays of immobilizedbiopolymers, a preselected site on the surface of the substrate isexposed to a solution of the desired biopolymer. In accordance with thepresent invention, this can be accomplished manually by applying analiquot of biopolymer solution to a preselected location on thesubstrate. Alternatively, thermal inkjet printing techniques utilizingcommercially available jet printers and piezoelectric microjet printingtechniques, as described in U.S. Pat. No. 4,877,745, can be utilized tospot selected substrate surface sites with selected biopolymers.

A wide variety of array formats may be employed in accordance with thepresent invention. One particularly useful format is a linear array ofnucleic acid probes, generally referred to in the art as a dipstick.Another suitable format comprises a two-dimensional pattern of discretecells. Of course, as would be readily appreciated by those skilled inthe art, other array formats would be equally suitable for use inaccordance with the present invention.

The array of the present invention may be a part of a variety ofdevices, such microtiter plates, test tubes, inorganic sheets,dipsticks, etc. For example, when the substrate is a thread, one or moreof such threads can be affixed to a plastic dipstick-type device. Whenthe substrate is in a form of a membrane, it can be affixed to glassslides. The particular device is, in and of itself, unimportant, as longas the substrate is securely affixed to the device without affecting thefunctional behavior of the substrate or any adsorbed biopolymer. Thedevice should also be stable to any materials into which the device isintroduced (e.g., clinical samples, etc.).

The method of making an assay article may further include a step ofexposing the assay article to a reagent, such as ammonium hydroxide,ethanol, or protein. In a preferred embodiment of this invention,ethanol is used for nucleic acid arrays and casein is used for proteinarrays.

Direct adsorption of biopolymers on aminated polypropylene substrates iswell-suited for use in the construction of genosensors and otherarray-based systems, such as differential gene expression micro-arrays.An aminated polypropylene substrate with the adsorbed biopolymers of thepresent invention may also be used as a device for performing a ligandbinding assay or for performing a hybridization assay by either reversehybridization (probes attached) or southern blot (target attached). Sucha device may also be used in an immunoassay.

Accordingly, another aspect of the present invention provides a methodof detecting a target biopolymer contained in a sample. The methodcomprises the steps of:

-   -   (a) providing an aminated substrate;    -   (b) providing a probe biopolymer that can form a complex with        the target biopolymer;    -   (c) contacting either the probe or target biopolymer with a        surface of the aminated substrate under a condition sufficient        for a direct adsorption of either the probe or target biopolymer        on the substrate surface to form a probe assay article or a        target assay article, respectively;    -   (d) contacting the probe assay article with the target        biopolymer, or contacting the target assay article with the        probe biopolymer under a condition that allows the formation of        a complex comprising the probe and the target biopolymers; and    -   (e) detecting and determining the presence of the complex as a        measurement for the presence or the amount of the target        biopolymer contained in the sample.

For the purpose of the present invention, probe biopolymer recognizesand binds to the target biopolymer forming a probe-target complex. Boththe probe and the target biopolymers may be selected from a groupconsisting of nucleic acids, polypeptides, proteins, and theiranalogues. For example, when the target is a polynucleotide, the probemay comprise a polynucleotide that is complimentary to the targetpolynucleotide (see FIG. 1). When the target is a receptor or a ligand,the probe may comprise a ligand or a receptor that respectivelyrecognizes and binds to the target receptor or ligand. When the targetis an antigen, the probe may comprise an antibody that recognizes theantigen, or vice versa (see FIG. 2).

Either target or probe may be directly adsorbed on the substrate. Forexample, in the Southern blot or Northern blot applications, targets areadsorbed on the substrate. Then, the substrate with the adsorbed targetsis contacted with the probes, preferably labeled, to detect the targetbiopolymers. To the contrary, in Ligand Binding assays or AffinityPurification assays, probes are bound to the substrate first. Then,target contained in a sample solution is contacted with the probesadsorbed on the substrate.

For the purpose of the present invention, an adsorption condition issufficient if the probe or target can adsorb on the substrate. Such acondition may vary, depending on the type of the biopolymers and theirsize. For instance, in one embodiment, described in detail in thefollowing Example 1, 10 nl aliquots of cDNA solutions are applied to anaminated polypropylene substrate. Following the application of the cDNA,the substrates are dried at 35° C. for 15 minutes. Then, the substratesare either soaked in ethanol for one hour or in ammonium hydroxide for15 minutes to remove loosely bound nucleic acid. Finally, the slides arebriefly rinsed with water and air-dried. One skilled in the art canreadily determine the suitable conditions for adsorbing other probes ortargets in view of the teaching of the present invention. As discussedabove, a variety of substrates may be used. In a preferred embodiment,however, aminated polypropylene substrates are used.

Contacting the probes with the targets (or hybridization) is conductedunder conditions that allow the formation of stable complexes betweenprobes and targets. For example, when target polynucleotides arecontacted with probe polynucleotides adsorbed on an aminatedpolypropylene substrate, complementary regions on the target and theprobe polynucleotides anneal to each other, forming probe-targetcomplex. The selection of such conditions is within the level of skillin the art and include those in which a low, substantially zero,percentage of mismatched hybrids form. The precise conditions depend,however, on the desired selectivity and sensitivity of the assay. Suchconditions include, but are not limited to, the hybridizationtemperature, the ionic strength and viscosity of the buffer, and therespective concentrations of the target and probe biomolecules.Hybridization conditions may be initially chosen to correspond to thoseknown to be suitable in standard procedures for hybridization to filtersand then optimized for use with the aminated polypropylene substrates ofthe present invention. The conditions suitable for hybridization of onetype of target material would appropriately be adjusted for use withother target materials.

For example, in certain embodiments the target polynucleotides arehybridized to the probe polynucleotides at temperatures in the range ofabout 20° C. to about 70° C., for a period from about 1 hour to about 24hours, in a suitable hybridization buffer. Suitable hybridizationbuffers for use in the practice of the present invention generallycontain a high concentration of salt. A typical hybridization buffercontains in the range of about 2× to about 6×SSC and about 0.01% toabout 0.5% SDS at pH 7-8. Once the probe/target complex is formed, thesubstrates are washed under conditions suitable to remove substantiallyall non-specifically bound target or probe biopolymers. Preferably, thewashing is carried out at a temperature in the range of about 20° C.-70°C. with a buffer containing about 0.1-6×SSC and 0.01-0.1% SDS. The mostpreferred wash conditions for polynucleotides presently include atemperature, which is the same as hybridization temperature, and abuffer containing 2×SSC and 0.01% SDS. As previously noted, it would bea routine matter for those working in the field to optimize thecontacting (hybridization) conditions for any given combination oftarget and probe biopolymers.

In accordance with embodiments of the present invention, either thetargets or probes of the present invention may be labeled with areporter. Delectability may be provided by such characteristics as colorchange, luminescence, fluorescence, or radioactivity. Examples ofreporters include, but are not limited to, dyes, chemiluminescentcompounds, enzymes, fluorescent compounds, metal complexes, magneticparticles, biotin, haptens, radio frequency transmitters, andradioluminescent compounds. One skilled in the art can readily determinethe type of reporter to be used once the type of target or probebiopolymers is determined.

The labeling procedure may occur prior to analysis (direct labeling) orafter hybridization (indirect labeling). An example of indirect labelingwould be the biotinylation of a target polynucleotide, hybridizing itwith a probe, and reacting the target-probe complexes with astreptavidin-alkaline phosphatase conjugate. The biotin moietiesretained after the hybridization with probe polynucleotides bind to astreptavidin-alkaline phosphatase conjugate, which then acts on achromogenic substrate, such as Enzyme Labeled Fluorescent (ELF) reagent.

For the purpose of the present invention, the same or differentbiopolymers may be attached to the substrates. If the biopolymers aredifferent, preferably they are located in isolated areas of thesubstrate to form arrays. For example, a substrate may be a microplate.Different biopolymers may be adsorbed within different wells of themicroplate for forming arrays. In accordance with another embodiment ofthe present invention, the substrate may be a slide and differentbiopolymers are adsorbed on different areas of the slide to form anarray.

The signal produced by an array may be detected by a naked eye or bymeans of a specially designed instrumentation, such as a confocal arrayreader. For example, in one embodiment, a fluorescent signal is recordedwith a charged coupled device (CCD) camera. It would be appreciated bythose skilled in the art, that the choice of a particular method used todetect and quantify the signal is not crucial for this invention.Essentially, any detection method may be used as long as it providesconsistent and accurate results.

Another aspect of the present invention provides an assay article fordetecting target biopolymers. The assay article of the present inventioncomprises a substrate and a biopolymer directly adsorbed on the surfaceof the substrate.

The substrate may be made of a variety of materials. In one embodimentthe substrate is made of polypropylene or polyethylene. Polypropyleneand polyethylene are organic materials that can be surface activated,but otherwise are chemically inert under harsh chemical conditions.Polypropylene can be used in very corrosive environments. For example,polypropylene has good chemical resistance to a variety of mineral acids(e.g., hydrochloric acid), organic acids (e.g., formic acid, aceticacid), bases (e.g., ammonium hydroxide, potassium hydroxide), salts(e.g., sodium chloride), oxidizing agents (e.g., peracetic acid, iodinesolutions), and organic solvents (e.g. acetone, ethyl alcohol,acetonitrile, dichloromethane, etc.). Additionally, polypropylene andpolyethylene are hydrophobic and provide a low fluorescence background.Amino groups may be introduced onto the polypropylene and polyethylenesurface by using a plasma discharge in an ammonia ororganic-amine-containing gas, as described above.

The assay article of the present invention may be molded into a varietyof shapes including, but not limited to, foams, filaments, threads,sheets, films, slides, gels, membranes, beads, plates, and likestructures.

The biopolymer may be selected from the group consisting of nucleicacids, polypeptides, proteins, and analogues thereof. A probe biopolymermay be a polynucleotide, such as an amplified DNA, cDNA, RNA, orprotein. A target biopolymer may be amplified DNA, cDNA,oligonucleotide, PNA, RNA or protein.

In a preferred embodiment, the substrate is made of an aminatedpolypropylene substrate in a form of a slide and the adsorbed biopolymeris a polynucleotide. Polynucleotides of various lengths may be directlyadsorbed on the aminated polypropylene substrate. According toembodiments of the present invention, the length of the adsorbedpolynucleotides may be from about 20 bp to about 10 kb. In order toachieve higher efficiency of adsorption, the length of the adsorbedpolynucleotides is preferably between about 100 bp and about 10 kb.

Another aspect of the present invention provides a test kit fordetecting a target biopolymer contained in a sample. The kit comprisesan aminated polypropylene substrate and a probe biopolymer directlyadsorbed on a surface of the substrate. When the probe polymer iscontacted with the target biopolymer, they form a complex that can bedetected by utilizing reporters and signal detection devices. The kitmay also include a reporter for generating a signal, which indicatesformation of the complex. Preferably, a plurality of the same ordifferent biopolymers are attached to the substrate forming an array.

According to the present invention and as it is described above, thesame or different biopolymers may be attached to the aminatedpolypropylene substrates to form an array.

The invention may be better understood with reference to theaccompanying example that is intended for purposes of illustration onlyand should not be construed as, in any sense, limiting the scope of thepresent invention, as defined in the claims appended hereto. While thedescribed procedures in the following example are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized indeed, those of ordinary skill in the art canreadily envision and produce further embodiments, based on the teachingsherein, without undue experimentation.

EXAMPLE 1

Preparation of cDNA Arrays

Polypropylene slides were surface aminated by a radio-frequencydischarge into ammonia gas, as described in Coassin et al. (U.S. Pat.No. 5,554,501, assigned to the assignee of the present invention).Unmodified cDNA of actin, β-microglobulin, G3PDH, p53, and TNF-a geneswere prepared each at a final concentration of 1M in 50 mM sodiumbicarbonate buffer, pH 9. A Biomek 2000™ robotic system (BeckmanCoulter, Inc., CA) equipped with a 384-pin HDRT system (Beckman Coulter,Inc., CA) was used to apply 10 nl aliquots of cDNA solutions onto theaminated polypropylene slides. A set of 5-(Biotinamido)pentylamine(BAPA) markers, (Pierce Chemical, Ill.), were also printed at both endsof each slide, thereby flanking the cDNAs. BAPA, which bindsstreptavidin-enzyme conjugate independently of hybridization, serves asan internal control for assay robustness. Following the application ofthe cDNA and BAPA markers, the slides were dried at 35° C. for 15minutes. Then the slides were either soaked in ethanol for one hour orin ammonium hydroxide for 15 minutes to remove loosely bound nucleicacid. Then, the slides were briefly rinsed with water and air-dried. Inone case, an ethanol-quenched slide was further rinsed in 1M NaOH for 15minutes. The slides were stored at −20° C. overnight.

Hybridization

For hybridization, each slide was brought to a room temperature anddenatured for 15 minutes in 0.15M NaCl and 0.5M NaOH solution. A mixtureof biotin-labeled cDNA targets of actin, β-microglobulin, G3PDH, and p53were applied at a final concentration of; 0.5 nM, following denaturationand the addition of hybridization buffer (2.4×SSC, 0.016% SDS, 0.28MTRIS, 0.028 NaCl, pH 7.5). The TNF-α target was not added so thatnon-specific hybridization could be measured to the TNF-α probe.Hybridization was allowed to proceed for 1 hour at 60° C., followed by astringency rinse in 2×SSC, 0.01% SDS at the same temperature. The slideswere incubated with streptavidin-alkaline phosphatase, and ELF reagent(fluorescent substrate for alkaline phosphatase) for 30 min. Followingthe incubation, the fluorescent signal image was recorded using a CCDcamera.

Results

An array of cDNA and BAPA markers were successfully adsorbed on theaminated polypropylene slides, as determined by hybridization of amixture of the corresponding cDNAs (FIG. 1). FIG. 1 shows Biomek HDRTprinting of cDNA and BAPA markers onto aminated polypropylene slides.The TNF-α signal was absent (negative control), while the remaining cDNAhybridization signals remained approximately at the same intensity asthe signals from the BAPA markers (positive control). The signal fromthe ammonium-hydroxide-quenched slide was significantly reduced in theintensity over that of the ethanol-quenched slides. There was nosignificant difference between ethanol- vs. ethanol-NaOH-pretreatedslides. This example illustrates that plasma aminated polypropyleneslides are capable of direct and stable adsorption of cDNA withoutchemical crosslinking.

Thus, the aminated polypropylene slides with directly adsorbedbiopolymers of the present invention and the method of their use indetection of target biopolymers are well adapted to attain all of theends and objects set forth above, together with other advantages, whichare inherent to the system. The present invention may be embodied inother specific forms without departing from its essentialcharacteristics. The described embodiment is to be considered in allrespects only as illustrative and not as restrictive. The scope of theinvention is, therefore, indicated by the appended claims rather than bythe foregoing description. All changes which come within the meaning andrange of the equivalence of the claims are to be embraced within theirscope.

EXAMPLE 2

Preparation of Protein Array

Human IgG attachment: Polypropylene film was surface aminated by aradio-frequency discharge as described in Example 1. Diluted Human IgG(Pierce Chemicals, cat. # 31877, 28 mg/mL) stock to 1 mg/mL in sodiumbicarbonate (50 mM, pH 9) and 4% sodium sulfate. Twenty-one 0.5 μL spotswere pipetted onto an amino polypropylene strip 2 cm wide and 8 cm long.Attachment reaction was allowed to proceed for 60 min. at 25° C. Thefilm was rinsed with Casein solution (1 mg/mL in 50 mM sodium carbonate,0.15M NaCl pH 9) for 60 min. at 25° C., and then rinsed twice indeionized water and by 1×TBS, 0.02% Tween-20, pH 7.4 briefly. The stripwas then used for binding assay.

Conjugation with goat anti-human IgG alkaline phosphatase: The 200 μL ofdiluted goat anti-human IgG alkaline phosphatase (Pierce Chemicals, cat.# 31310) solution 1:1000 in blocking buffer (1×TBS, 1 mg/mL Casein,0.02% Tween-20 pH 7.4) was pipetted to a petri dish and theabove-spotted polypropylene strip was placed on it. Spotted side of thepolypropylene strip down, on top of the solution and was incubated for60 min. at 25° C. The polypropylene strip was then rinsed 4 times in 20mL of 1×TBS, 0.02% Tween-20, pH 7.4.

ELF detection: To detect the fluorescence signal, the enzyme substrate,ELF, was prepared by mixing components A and B (1:25) (Molecular Probes,Eugene, Oreg.) and 200 μL solution for each strip was used as describedabove. After 30 min. incubation at 22° C., the strip was dipped once indeionized water. The signals were detected using 365 nm UV light and aCCD camera, having 520 nm filter as shown in FIG. 2.

Results

An array of IgG was successfully adsorbed on the aminated polypropylenefilm as shown in FIG. 2.

Thus, the aminated polypropylene slides with directly adsorbedbiopolymers of the present invention and the method of their use indetection of target biopolymers are well adapted to attain all of theends and objects set forth above, together with other advantages, whichare inherent to the system. The present invention may be embodied inother specific forms without departing from its essentialcharacteristics. The described embodiment is to be considered in allrespects only as illustrative and not as restrictive. The scope of theinvention is, therefore, indicated by the appended claims rather than bythe foregoing description. All changes which come within the meaning andrange of the equivalence of the claims are to be embraced within theirscope.

1. A method of making an assay article for use in biopolymer detectioncomprising the steps of: (a) providing a biopolymer other than anadhesive protein; (b) modifying a surface of a substrate to obtain amodified surface having physical properties that promote non-covalentbinding of the biopolymer to the substrate; and (c) contacting thebiopolymer with the modified surface of the substrate and air-drying thesubstrate whereby the biopolymer immobilizes on the modified surface ofthe substrate without additional fixing steps and without chemicalcrosslinking.
 2. The method of claim 1, wherein the biopolymer isunmodified prior to the contacting step.
 3. The method of claim 1,wherein the biopolymer is modified prior to the contacting step.
 4. Themethod of claim 1, wherein the substrate is selected from the groupconsisting of organic polymers, their analogs, blends and copolymersselected from the group consisting of polycarbonates, polyethylenes,polypropylenes, polymethacrylates, polymethylpentenes, polysulfones,polytetrafluoroethylenes, and polyvinylidene difluorides.
 5. The methodof claim 4, wherein modified substrate is aminated polypropylene.
 6. Themethod of claim 1, wherein the substrate is in a form selected from thegroup consisting of foams, filaments, threads, sheets, films, slides,gels, membranes, beads, plates, and like structures.
 7. The method ofclaim 1, wherein the contacting step is carried out by a techniqueselected from a group consisting of jet printing, solid or opencapillary device contact printing, microfluidic channel printing, silkscreening, printing using devices based upon electrochemical orelectromagnetic forces, and manual spotting.
 8. The method of claim 1,wherein the biopolymer is selected from a group consisting of nucleicacids, polypeptides, proteins, and analogues thereof.
 9. The method ofclaim 8, wherein the biopolymer is a polynucleotide.
 10. The method ofclaim 9, wherein the polynucleotide is cDNA.
 11. The method of claim 1,wherein the step of providing the biopolymer comprises providing asolution of the biopolymer and the step of contacting comprises: (a)placing an aliquot of the biopolymer solution on the modified substrate;and (b) air-drying the substrate to directly adsorb the biopolymer onthe surface of the substrate.
 12. The method of claim 11, wherein themodified substrate is an amino-modified substrate.
 13. The method ofclaim 12, wherein the amino-modified substrate is aminopolypropylene.14. The method of claim 11, wherein the amount of the biopolymer appliedto the substrate ranges from about 10⁻²⁰ to about 10⁻¹⁴ moles.
 15. Themethod of claim 14, wherein the biopolymer is a polynucleotide, and theamount of polynucleotide is about 10⁻¹⁸ moles.
 16. The method of claim11, wherein the aliquot is from about 0.1 nL to about 500 nL.
 17. Themethod of claim 16, wherein the biopolymer is a polynucleotide, and thealiquot is about 10 nL.
 18. The method of claim 11, wherein theair-drying step is conducted for a period ranging from about 5 minutesto about 60 minutes.
 19. The method of claim 18, wherein the air-dryingstep is conducted for a period of about 15 min.
 20. The method of claim11, wherein a plurality of the biopolymers are placed and adsorbed onthe surface of the modified substrate in an array.
 21. The method ofclaim 20, wherein the modified substrate is an amino-modified substrate.22. The method of claim 21, wherein the amino-modified substrate is anamino propylene.
 23. The method of claim 11, further comprising a stepof washing the assay article with a reagent.
 24. The method of claim 23,wherein the reagent is selected from a group consisting of ammoniumhydroxide, ethanol, and protein.
 25. The method of claim 24, wherein theprotein is casein.
 26. The method of claim 1, wherein the substrate ismade of polypropylene or polyethylene.
 27. The method of claim 26,further comprising a step of modifying the substrate surface prior tothe contacting step, wherein the step of modifying the substratecomprises introduction of a functionality selected from a groupconsisting of amino, carboxyl, hydroxyl, thiol, and their derivatives.28. The method of claim 27, wherein the functionality is an amino group.29-42. (canceled)
 43. An assay article, comprising: a non-polystyrenesubstrate having a functionality selected from a group consisting ofamino, carboxyl, hydroxyl, thiol and their derivatives, and a biopolymerimmobilized on a surface of the substrate without forming a covalentbond with the substrate.
 44. The assay article of claim 43, wherein thesubstrate is in a form selected from the group consisting of foams,filaments, threads, sheets, films, slides, gels, membranes, beads,plates, and planar devices having discrete isolated areas in the form ofwells, troughs, pedestals, hydrophobic or hydrophilic patches, die-cutadhesive reservoirs, or other physical barriers to fluid flow.
 45. Theassay article of claim 43, wherein the biopolymer is selected from agroup consisting of nucleic acids, polypeptides, proteins, and analoguesthereof.
 46. The assay article of claim 45, wherein the biopolymer is aprotein or a polynucleotide.
 47. (canceled)
 48. The assay article ofclaim 43, wherein the substrate is aminated.
 49. The assay article ofclaim 43, wherein the biopolymer is polynucleotide and the substrate isin a form of a slide.
 50. The assay article of claim 49, wherein thelength of the polynucleotide is in a range from about 20 bp to about 10kb.
 51. The assay article of claim 43, wherein a plurality of the sameor different biopolymers are attached to discrete, isolated areas of thesubstrate surface by direct adsorption to form an array.
 52. A test kitfor detecting a target biopolymer contained in a sample comprising: anaminated non-polystyrene substrate; and a probe biopolymer immobilizedon a surface of the substrate without forming a covalent bond with thesubstrate, wherein the probe biopolymer forms a complex with the targetbiopolymer.
 53. The test kit of claim 52 further comprising a reporterselected from the group consisting of dyes, chemiluminescent compounds,enzymes, fluorescent compounds, metal complexes, magnetic particles,biotin, haptens, radio frequency transmitters, and radioluminescentcompounds.
 54. The test kit of claim 53, wherein a plurality of the sameor different probe biopolymers are attached to discrete, isolated areasof the substrate surface by direct adsorption forming an array. 55-71.(canceled)
 72. The assay article of claim 43, wherein the biopolymer isimmobilized by direct adsorption on the surface of the substrate. 73.The assay article of claim 52, wherein the biopolymer is immobilized bydirect adsorption on the surface of the substrate.
 74. The method ofclaim 1 further comprising washing the assay article to remove looselybound biopolymers, wherein the washing is conducted immediately afterthe drying step is completed.
 75. An assay article prepared by themethod of claim
 1. 76. The assay article of claim 43, wherein thesubstrate is of polypropylene, polyethylene, or both.
 77. The test kitof claim 52, wherein the substrate is of aminated polypropylene.